Members of the microRNA-17-92 cluster exhibit a cell-intrinsic antiangiogenic function in endothelial cells

Abstract MicroRNAs are endogenously expressed small noncoding RNAs that regulate gene expression on the posttranscriptional level. The miR-17-92 cluster (encoding miR-17, -18a, -19a/b, -20a, and miR-92a) is highly expressed in tumor cells and is up-regulated by ischemia. Whereas miR-92a was recently identified as negative regulator of angiogenesis, the specific functions of the other members of the cluster are less clear. Here we demonstrate that overexpression of miR-17, -18a, -19a, and -20a significantly inhibited 3-dimensional spheroid sprouting in vitro, whereas inhibition of miR-17, -18a, and -20a augmented endothelial cell sprout formation. Inhibition of miR-17 and miR-20a in vivo using antagomirs significantly increased the number of perfused vessels in Matrigel plugs, whereas antagomirs that specifically target miR-18a and miR-19a were less effective. However, systemic inhibition of miR-17/20 did not affect tumor angiogenesis. Further mechanistic studies showed that miR-17/20 targets several proangiogenic genes. Specifically, Janus kinase 1 was shown to be a direct target of miR-17. In summary, we show that miR-17/20 exhibit a cell-intrinsic antiangiogenic activity in endothelial cells. Inhibition of miR-17/20 specifically augmented neovascularization of Matrigel plugs but did not affect tumor angiogenesis indicating a context-dependent regulation of angiogenesis by miR-17/20 in vivo.

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Genetic alteration of endothelial heparan sulfate selectively inhibits tumor angiogenesis

The Journal of Cell Biology ◽

10.1083/jcb.200610086 ◽

2007 ◽

Vol 177 (3) ◽

pp. 539-549 ◽

Cited By ~ 85

Author(s):

Mark M. Fuster ◽

Lianchun Wang ◽

Janice Castagnola ◽

Lyudmila Sikora ◽

Krisanavane Reddi ◽

...

Keyword(s):

Endothelial Cells ◽

Heparan Sulfate ◽

Tumor Angiogenesis ◽

Wound Repair ◽

Reproductive Capacity ◽

Erk Phosphorylation ◽

Tumor Endothelium ◽

A Cell

To examine the role of endothelial heparan sulfate during angiogenesis, we generated mice bearing an endothelial-targeted deletion in the biosynthetic enzyme N-acetylglucosamine N-deacetylase/N-sulfotransferase 1 (Ndst1). Physiological angiogenesis during cutaneous wound repair was unaffected, as was growth and reproductive capacity of the mice. In contrast, pathological angiogenesis in experimental tumors was altered, resulting in smaller tumors and reduced microvascular density and branching. To simulate the angiogenic environment of the tumor, endothelial cells were isolated and propagated in vitro with proangiogenic growth factors. Binding of FGF-2 and VEGF164 to cells and to purified heparan sulfate was dramatically reduced. Mutant endothelial cells also exhibited altered sprouting responses to FGF-2 and VEGF164, reduced Erk phosphorylation, and an increase in apoptosis in branching assays. Corresponding changes in growth factor binding to tumor endothelium and apoptosis were also observed in vivo. These findings demonstrate a cell-autonomous effect of heparan sulfate on endothelial cell growth in the context of tumor angiogenesis.

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The recombinant lectin-like domain of thrombomodulin inhibits angiogenesis through interaction with Lewis Y antigen

Blood ◽

10.1182/blood-2011-08-376038 ◽

2012 ◽

Vol 119 (5) ◽

pp. 1302-1313 ◽

Cited By ~ 31

Author(s):

Cheng-Hsiang Kuo ◽

Po-Ku Chen ◽

Bi-Ing Chang ◽

Meng-Chen Sung ◽

Chung-Sheng Shi ◽

...

Keyword(s):

Endothelial Cells ◽

Egf Receptor ◽

Tube Formation ◽

Endothelial Tube Formation ◽

A Cell ◽

Lewis Y ◽

Tm Domain

AbstractLewis Y Ag (LeY) is a cell-surface tetrasaccharide that participates in angiogenesis. Recently, we demonstrated that LeY is a specific ligand of the recombinant lectin-like domain of thrombomodulin (TM). However, the biologic function of interaction between LeY and TM in endothelial cells has never been investigated. Therefore, the role of LeY in tube formation and the role of the recombinant lectin-like domain of TM—TM domain 1 (rTMD1)—in antiangiogenesis were investigated. The recombinant TM ectodomain exhibited lower angiogenic activity than did the recombinant TM domains 2 and 3. rTMD1 interacted with soluble LeY and membrane-bound LeY and inhibited soluble LeY-mediated chemotaxis of endothelial cells. LeY was highly expressed on membrane ruffles and protrusions during tube formation on Matrigel. Blockade of LeY with rTMD1 or Ab against LeY inhibited endothelial tube formation in vitro. Epidermal growth factor (EGF) receptor in HUVECs was LeY modified. rTMD1 inhibited EGF receptor signaling, chemotaxis, and tube formation in vitro, and EGF-mediated angiogenesis and tumor angiogenesis in vivo. We concluded that LeY is involved in vascular endothelial tube formation and rTMD1 inhibits angiogenesis via interaction with LeY. Administration of rTMD1 or recombinant adeno-associated virus vector carrying TMD1 could be a promising antiangiogenesis strategy.

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BMX Represses Thrombin-PAR1–Mediated Endothelial Permeability and Vascular Leakage During Early Sepsis

Circulation Research ◽

10.1161/circresaha.119.315769 ◽

2020 ◽

Vol 126 (4) ◽

pp. 471-485 ◽

Cited By ~ 5

Author(s):

Zhao Li ◽

Mingzhu Yin ◽

Haifeng Zhang ◽

Weiming Ni ◽

Richard W. Pierce ◽

...

Keyword(s):

Endothelial Cells ◽

Cecal Ligation ◽

Endothelial Permeability ◽

Vascular Leakage ◽

Lung Epithelial Cells ◽

Negative Regulator ◽

Cecal Ligation And Puncture ◽

Early Sepsis

Rationale: BMX (bone marrow kinase on the X chromosome) is highly expressed in the arterial endothelium from the embryonic stage to the adult stage in mice. It is also expressed in microvessels and the lymphatics in response to pathological stimuli. However, its role in endothelial permeability and sepsis remains unknown. Objective: We aimed to delineate the function of BMX in thrombin-mediated endothelial permeability and the vascular leakage that occurs with sepsis in cecal ligation and puncture models. Methods and Results: The cecal ligation and puncture model was applied to WT (wild type) and BMX-KO (BMX global knockout) mice to induce sepsis. Meanwhile, the electric cell-substrate impedance sensing assay was used to detect transendothelial electrical resistance in vitro and, the modified Miles assay was used to evaluate vascular leakage in vivo. We showed that BMX loss caused lung injury and inflammation in early cecal ligation and puncture–induced sepsis. Disruption of BMX increased thrombin-mediated permeability in mice and cultured endothelial cells by 2- to 3-fold. The expression of BMX in macrophages, neutrophils, platelets, and lung epithelial cells was undetectable compared with that in endothelial cells, indicating that endothelium dysfunction, rather than leukocyte and platelet dysfunction, was involved in vascular permeability and sepsis. Mechanistically, biochemical and cellular analyses demonstrated that BMX specifically repressed thrombin-PAR1 (protease-activated receptor-1) signaling in endothelial cells by directly phosphorylating PAR1 and promoting its internalization and deactivation. Importantly, pretreatment with the selective PAR1 antagonist SCH79797 rescued BMX loss-mediated endothelial permeability and pulmonary leakage in early cecal ligation and puncture–induced sepsis. Conclusions: Acting as a negative regulator of PAR1, BMX promotes PAR1 internalization and signal inactivation through PAR1 phosphorylation. Moreover, BMX-mediated PAR1 internalization attenuates endothelial permeability to protect vascular leakage during early sepsis.

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Carcinoembryonic Antigen (CEA) Regulates Tumor-Angiogenesis in Colon Carcinomas.

Blood ◽

10.1182/blood.v112.11.1897.1897 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1897-1897

Author(s):

Kira Braemswig ◽

Marina Poettler ◽

Wazlawa Kalinowska ◽

Christoph Zielinski ◽

Gerald W Prager

Keyword(s):

Cell Proliferation ◽

Endothelial Cells ◽

Endothelial Cell ◽

Tumor Angiogenesis ◽

Endothelial Cell Proliferation ◽

Erk Phosphorylation ◽

Dose Dependent Increase ◽

Dose Dependent

Abstract Human carcinoembryonic antigen (CEA) is a cell surface adhesion molecule member of the Immunoglobulin Superfamily (IgSF). Aberrant upregulation and secretion of soluble CEA is a common feature found in a wide variety of human cancers such as colon, breast and lung. Previous in vitro and in vivo results have demonstrated that CEA can affect tumor cell behavior including the inhibition of cell differentiation and apoptosis. However, any functional effects on angiogenic endothelial cell behavior are so far unknown. In the present work we found that in endothelial cells exogenous CEA led to a time and dose dependent increase in ERK phosphorylation, which was inhibited by the specific MEK inhibitor U0126. Thereby, the observed CEA effect was comparable in time and intense with the canonical angiogenic growth factor VEGF. The CEA-induced ERK phosphorylation was not affected by the blockage of VEGFR-2 / flk-1 using a specific inhibiting peptide (CBO-P11), which indicates a VEGF-independent mechanism. Furthermore, co-stimulation of endothelial cells with VEGF and CEA shows synergistic effects on ERK phosphorylation. While in endothelial cells no endogenous expression of CEA is detected, its putative receptor, the CEA receptor (CEAR), is highly expressed as shown by immunohistochemical staining of paraffin-embedded colon carcinoma sections as well as in biochemical analyses. When an activating antibody against CEAR was used, CEA-induced ERK phosphorylation was mimicked, while downregulation of CEAR by siRNA diminished CEA-induced signal transduction, significantly. To test a biological relevance of our findings, we first measured endothelial cell proliferation: CEA led to a dose dependent increase in endothelial cell proliferation in vitro, which again revealed a synergistic effect with VEGF. Thereby, CEA-induced endothelial cell proliferation was again independent of VEGFR-2 / flk-1. A biological role of CEA in tumor-angiogenesis was reflected by an in vivo model using CEA Mimotope immunized BALB/c mice, which were transplanted with MethA/CEA overexpressing tumor cells. Immunohistological analyses of these tumors revealed a significantly reduced vascular density, which was accompanied with diminished tumor growth. Our data provide first evidence of CEA as a novel pro-angiogenic activator of endothelial cells, which results in an increase in endothelial cell proliferation, independent of VEGFR-2. Furthermore, by targeting CEA in an in vivo mouse model, tumor-angiogenesis was markley reduced, indicating a potential therapeutic target in cancer.

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Human Erythropoietin Induces a Pro-Angiogenic Phenotype in Cultured Endothelial Cells and Stimulates Neovascularization In Vivo

Blood ◽

10.1182/blood.v93.8.2627 ◽

1999 ◽

Vol 93 (8) ◽

pp. 2627-2636 ◽

Cited By ~ 376

Author(s):

Domenico Ribatti ◽

Marco Presta ◽

Angelo Vacca ◽

Roberto Ria ◽

Roberta Giuliani ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Chorioallantoic Membrane ◽

Janus Kinase ◽

Angiogenic Factor ◽

Matrix Metalloproteinase 2 ◽

Angiogenic Potential ◽

Angiogenic Response

Abstract Hematopoietic and endothelial cell lineages share common progenitors. Accordingly, cytokines formerly thought to be specific for the hematopoietic system have been shown to affect several functions in endothelial cells, including angiogenesis. In this study, we investigated the angiogenic potential of erythropoietin (Epo), the main hormone regulating proliferation, differentiation, and survival of erythroid cells. Epo receptors (EpoRs) have been identified in the human EA.hy926 endothelial cell line by Western blot analysis. Also, recombinant human Epo (rHuEpo) stimulates Janus Kinase-2 (JAK-2) phosphorylation, cell proliferation, and matrix metalloproteinase-2 (MMP-2) production in EA.hy926 cells and significantly enhances their differentiation into vascular structures when seeded on Matrigel. In vivo, rHuEpo induces a potent angiogenic response in the chick embryo chorioallantoic membrane (CAM). Accordingly, endothelial cells of the CAM vasculature express EpoRs, as shown by immunostaining with an anti-EpoR antibody. The angiogenic response of CAM blood vessels to rHuEpo was comparable to that elicited by the prototypic angiogenic cytokine basic fibroblast growth factor (FGF2), it occurred in the absence of a significant mononuclear cell infiltrate, and it was not mimicked by endothelin-1 (ET-1) treatment. Taken together, these data demonstrate the ability of Epo to interact directly with endothelial cells and to elicit an angiogenic response in vitro and in vivo and thus act as a bona fide direct angiogenic factor.

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Decrease of miR-19b-3p in Brain Microvascular Endothelial Cells Attenuates Meningitic Escherichia coli-Induced Neuroinflammation via TNFAIP3-Mediated NF-κB Inhibition

Pathogens ◽

10.3390/pathogens8040268 ◽

2019 ◽

Vol 8 (4) ◽

pp. 268 ◽

Cited By ~ 2

Author(s):

Amjad ◽

Yang ◽

Li ◽

Fu ◽

Yang ◽

...

Keyword(s):

Escherichia Coli ◽

Endothelial Cells ◽

Negative Regulator ◽

Microvascular Endothelial Cells ◽

Brain Microvascular Endothelial Cells ◽

E Coli ◽

Inflammatory Damage ◽

Microvascular Endothelial

Meningitic Escherichia coli can traverse the host’s blood–brain barrier (BBB) and induce severe neuroinflammatory damage to the central nervous system (CNS). During this process, the host needs to reasonably balance the battle between bacteria and brain microvascular endothelial cells (BMECs) to minimize inflammatory damage, but this quenching of neuroinflammatory responses at the BBB is unclear. MicroRNAs (miRNAs) are widely recognized as key negative regulators in many pathophysiological processes, including inflammatory responses. Our previous transcriptome sequencing revealed numbers of differential miRNAs in BMECs upon meningitic E. coli infection; we next sought to explore whether and how these miRNAs worked to modulate neuroinflammatory responses at meningitic E. coli entry of the BBB. Here, we demonstrated in vivo and in vitro that meningitic E. coli infection of BMECs significantly downregulated miR-19b-3p, which led to attenuated production of proinflammatory cytokines and chemokines via increasing the expression of TNFAIP3, a negative regulator of NF-κB signaling. Moreover, in vivo injection of miR-19b-3p mimics during meningitic E. coli challenge further aggravated the inflammatory damage to mice brains. These in vivo and in vitro findings indicate a novel quenching mechanism of the host by attenuating miR-19b-3p/TNFAIP3/NF-κB signaling in BMECs in response to meningitic E. coli, thus preventing CNS from further neuroinflammatory damage.

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Hyperproliferative apoptosis-resistant endothelial cells in idiopathic pulmonary arterial hypertension

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00428.2006 ◽

2007 ◽

Vol 293 (3) ◽

pp. L548-L554 ◽

Cited By ~ 224

Author(s):

Fares A. Masri ◽

Weiling Xu ◽

Suzy A. A. Comhair ◽

Kewal Asosingh ◽

Michelle Koo ◽

...

Keyword(s):

Endothelial Cells ◽

Pulmonary Arterial Hypertension ◽

Arterial Hypertension ◽

Idiopathic Pulmonary Arterial Hypertension ◽

Janus Kinase ◽

Vascular Lesions ◽

Nuclear Antigen ◽

Pulmonary Arterial

Idiopathic pulmonary arterial hypertension (IPAH) is characterized by plexiform vascular lesions, which are hypothesized to arise from deregulated growth of pulmonary artery endothelial cells (PAEC). Here, functional and molecular differences among PAEC derived from IPAH and control human lungs were evaluated. Compared with control cells, IPAH PAEC had greater cell numbers in response to growth factors in culture due to increased proliferation as determined by bromodeoxyuridine incorporation and Ki67 nuclear antigen expression and decreased apoptosis as determined by caspase-3 activation and TdT-mediated dUTP nick end labeling assay. IPAH cells had greater migration than control cells but less organized tube formation in in vitro angiogenesis assay. Persistent activation of signal transducer and activator of transcription 3 (STAT3), a regulator of cell survival and angiogenesis, and increased expression of its downstream prosurvival target, Mcl-1, were identified in IPAH PAEC. A Janus kinase (JAK) selective inhibitor reduced STAT3 activation and blocked proliferation of IPAH cells. Phosphorylated STAT3 was detected in endothelial cells of IPAH lesions in vivo, suggesting that STAT3 activation plays a role in the proliferative pulmonary vascular lesions in IPAH lungs.

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ZNF191 Inhibits Hepatocellular Carcinoma Metastasis via DLG1-mediated YAP1 Inactivation

10.1101/027060 ◽

2015 ◽

Author(s):

Di Wu ◽

Guoyuan Liu ◽

Songmin Jiang ◽

Long Yu

Keyword(s):

Hepatocellular Carcinoma ◽

Zinc Finger Protein ◽

Negative Regulator ◽

Metastasis Suppressor ◽

Discs Large ◽

Chromatin Immunoprecipitation Sequencing ◽

A Cell ◽

Carcinoma Metastasis

Searching targets for hepatocellular carcinoma (HCC) treatment, we identified zinc finger protein 191 (ZNF191) as a suppressor against HCC metastasis. Over-expressing ZNF191 in HCC cells impaired cell motility, while ZNF191 depletion promoted HCC cell migration in vitro and metastasis in vivo through triggering yes-associated protein 1 (YAP1) signaling. Chromatin immunoprecipitation-sequencing (ChIP-seq) revealed that ZNF191 specifically bound to the promoter of Discs, Large homolog 1 (DLG1), a cell polarity maintainer and a negative regulator of YAP1. Double-knockdown experiments showed that DLG1 was not only the mediator of ZNF191 s function to suppress migration but also a link between ZNF191 and YAP1 signaling. ZNF191 was down-regulated in metastatic HCCs, correlating positively with DLG1 levels and inversely with YAP1 activation. Our findings indicate ZNF191 functions as a metastasis suppressor via DLG1-mediated YAP1 signaling inactivation.

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Angiomotin

The Journal of Cell Biology ◽

10.1083/jcb.152.6.1247 ◽

2001 ◽

Vol 152 (6) ◽

pp. 1247-1254 ◽

Cited By ~ 254

Author(s):

Boris Troyanovsky ◽

Tetyana Levchenko ◽

Göran Månsson ◽

Olga Matvijenko ◽

Lars Holmgren

Keyword(s):

Endothelial Cells ◽

Cell Migration ◽

Leading Edge ◽

Tube Formation ◽

Endothelial Cell Migration ◽

Kringle Domains ◽

A Cell ◽

Cell Type Specific

Angiostatin, a circulating inhibitor of angiogenesis, was identified by its ability to maintain dormancy of established metastases in vivo. In vitro, angiostatin inhibits endothelial cell migration, proliferation, and tube formation, and induces apoptosis in a cell type–specific manner. We have used a construct encoding the kringle domains 1–4 of angiostatin to screen a placenta yeast two-hybrid cDNA library for angiostatin-binding peptides. Here we report the identification of angiomotin, a novel protein that mediates angiostatin inhibition of migration and tube formation of endothelial cells. In vivo, angiomotin is expressed in the endothelial cells of capillaries as well as larger vessels of the human placenta. Upon expression of angiomotin in HeLa cells, angiomotin bound and internalized fluorescein-labeled angiostatin. Transfected angiomotin as well as endogenous angiomotin protein were localized to the leading edge of migrating endothelial cells. Expression of angiomotin in endothelial cells resulted in increased cell migration, suggesting a stimulatory role of angiomotin in cell motility. However, treatment with angiostatin inhibited migration and tube formation in angiomotin-expressing cells but not in control cells. These findings indicate that angiostatin inhibits cell migration by interfering with angiomotin activity in endothelial cells.

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